The Two Process model of homeostatic sleep regulation posits that the timing of sleep is due to the interaction between a circadian process (Process C) and a homeostatically regulated sleep process (Process S). Whereas Process C is linked to the suprachiasmatic nucleus, the neural basis of Process S is currently unknown. During the previous funding period, we identified a population of cells in the cerebral cortex that express the neuronal nitric oxide synthase (nNos) within which the transcription factor Fos is expressed specifically during recovery sleep (RS) after sleep deprivation (SD). Expression of Fos in cortical nNos neurons also occurs in conjunction with the natural occurrence of sleep. Based on these results, the hypothesis that underlies this proposal is that cortical Fos+/nNos neurons are fundamentally related to the homeostatic regulation of sleep and are a neuroanatomical substrate of Process S. To test this hypothesis, we will systematically vary the duration of SD and RS and determine Fos expression in cortical nNos neurons by double-label immunohistochemistry. To determine whether nNos per se is related to homeostatic sleep regulation, we will evaluate homeostatic sleep regulation in the nNos knockout mouse and use a co-localized phenotypic marker to determine whether Fos is induced during RS in cortical neurons that normally express nNos. To determine the afferent inputs that influence cortical nNos neurons, we will use a combination of in vitro and in vivo approaches. We will use patch clamp electrophysiology of cortical nNos/EGFP neurons to determine the neurotransmitters and neuromodulators that excite or inhibit these cells, particularly substances implicated in the control of sleep and wakefulness. Lastly, we will determine the role(s) of cholinergic, noradrenergic, histaminergic, and hypocretinergic inputs in state-specific cortical Fos+/nNos expression by elimination of these putative afferent populations using neurochemically-specific saporin conjugates. Information from these in vivo experiments will complement the in vitro studies and is essential to understand the neural circuitry that results in state-specific activation of nNos neurons. These experiments will elucidate the neurobiology of cortical nNos neurons and allow us to determine whether the activity of these cells is indeed related to Process S. The results will not only enhance our understanding of sleep/wake regulation, but may also have implications for understanding sleep disorders and neurological and psychiatric diseases involving the cerebral cortex such as epilepsy, anxiety, and schizophrenia. PUBLIC HEALTH RELEVANCE: During the previous funding period, we identified a population of cells in the cerebral cortex that express the neuronal nitric oxide synthase (nNos) that appear to be activated during sleep. This is the first neuronal population in the cerebral cortex known to be active during sleep. We will determine whether nNos itself is important for sleep or whether nNos is simply a marker for these cells. We will also identify the neurotransmitters and neuromodulators that activate or inhibit nNos neurons and which may therefore activate these cells during sleep and inhibit nNos neurons during wakefulness. The results will not only enhance our understanding of sleep/wake regulation, but may also have implications for understanding sleep disorders and neurological and psychiatric diseases involving the cerebral cortex such as epilepsy, anxiety and schizophrenia.